1. Field of the Invention
This invention is in the fields of chemistry and biology and provides a fluoroassay method for use therein. More particularly, it concerns a group of aryl substituted pyridine compounds and their use as components of or as intermediates toward components of fluorescent chelate labels for fluoroassay techniques
2. Background
Fluoroassay techniques are finding increasing application in chemical, biochemical and medical analyses. Fluorescence measurement methods are intrinsically extremely sensitive However, the sensitivity of fluorescence assays, in practice, is limited by the presence of background fluorescence.
U.S. Pat. Nos. 4,150,295 and 4,058,732, issued on Apr. 17, 1979 and Nov. 15, 1977, respectively, and a chapter appearing at pages 67-80 of Immunofluorescence and Related Staining Techniques, Knapp, et al eds. (1978, Elsevier/North Holland Biomedical Press) disclose the general concept that background fluorescence has a relatively short life and that one can advantageously employ as measured fluorescent species, materials having a longer lived fluorescence. This work further points out that by using an intermittent excitation source and a timecoupled measurement of fluorescence one could essentially avoid or reject the background fluorescence while measuring the desired fluorescence.
Rare earth chelates have been identified by the art as materials having long-lived fluorescence. Such materials include a rare earth metal ion such as terbium or europium chelated by one or more ligands such as amine polyacids (See, U.S. Pat. No. 4,352,751 of Wieder and Wollenberg), "heteroatom-containing groups" including iminodiacetate substituted phenols, coumarins and phenanthrolines (See, Eastman Kodak European patent application No. 0068875) and aromatic diketones (See, German OLS No. 2,628,158), to point out a number of representative disclosures.
The art recognizes that the following properties are desirable in a chelating group.
1. It should form a stable chelate complex with the rare earth--i.e., with a stability constant (log K) of 17 or greater.
2. The fluorescent chelate complex with rare earth should have a long-lived fluorescence, that is, a fluorescence that is not appreciably decayed when background interference has already decayed.
3. The fluorescence excitation should occur at as long a wavelength as possible--preferably at 300 nm or greater so as to avoid interference which commonly occurs in biological samples at wavelengths of about 270 nm.
4. The fluorescent complex should have an intense emission--i.e. it should have a high quantum yield.
In addition, the materials should have solubility, chemical stability and other properties that are compatible with the nature of the samples (usually aqueous samples) with which the materials are used.
Notwithstanding this recognition in the art, the materials described heretofore generally have been at least somewhat lacking in one or more of these properties. For example, EPO No. 0068875 points out that the fluorescence of reagents such as aromatic diketones (German OLS No. 2,628,158) is quenched in water because of an "aqueous stability" problem while the phenolic aromatic ketones, coumarins and phenanthrolines (which EPO No. 0068875 specifically discloses), have exhibited a lack of chemical stability and a low quantum yield. As a result of these deficiencies no one has yet produced a widely applicable reagent system for fluoroassay techniques.
What is needed is a fluorescent chelate system that better satisfies these properties.
The present invention concerns a family of substituted pyridine derivatives as well as intermediates thereto which may be incorporated into longlived fluorescent species with rare earth metals. References to the general class of substituted pyridine derivatives include U.S. Pat. Nos. 4,008,239, 3,970,662 and 3,907,808 as well as Carbeteas and Williams, J. Heterocycl. Chem., 11(5), 819 (1974); Weller and Luellen, Tetrahedron Letters, Vol. 22, No. 44, pp 4381-84 (1981) and Weller, Luellen and Weller J. Org. Chem., 47, 4803-06 (1982). The above-noted European patent application No. 0068875 is also of interest.